Nucleic acid molecule encoding a novel estrogen receptor beta variant

ABSTRACT

This invention relates to an isolated nucleotide fragment of a novel estrogen receptor, in particular, a novel ERβ variant protein and isolated nucleic acid fragment comprising the coding regions of the genes encoding such variant proteins. Also provided are vectors, host cells, and methods for producing the novel ERβ variant protein. The invention further relates to method of obtaining such nucleotide fragment and the method of determining the presence of such ERβ variant protein in a sample.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to a newly identified polynucleotide encoding anovel estrogen receptor beta variant, and polypeptide encoded by suchpolynucleotide, are potentially useful in therapeutic modulation ofpathophysiologic estrogen signaling.

BACKGROUND OF THE INVENTION

Estrogens are major regulators of many physiological functions, such asthose associated with reproduction and mammary gland development (Georgeet al. The physiology of Reproduction, 1988; Vol. I, page 3., RavenPress, New York). Estrogens influence growth, differentiation, andfunction of many target tissues including tissues of female and malereproductive tract. Estrogens also play an important role in themaintenance of bone mass and in the cardiovascular system whereestrogens have certain protective effects (Farhat et al. FASEB J. 1996;10:615-624). The effects of estrogen are mediated in either normal cellor neoplastic target cells via an initial interaction with the estrogenreceptor. Estrogen receptors (alpha (ERα) and beta (ERβ)) belong to anuclear hormone receptor superfamily of transcription factors (Green etal. Nature 1986; 320:134-139, Kuiper et al. Proc. Natl. Acad. Sci. USA.1996; 93(12):5925-30, Mosselman et al. FEBS Lett 1996 Aug. 19;392(1):49-53, Enmark et al. J. Clin. Endocrinol. Metab. 1997;82(12):4258-65, Bhat et al. J Steroid Biochem Mol Biol 1998;67(3):233-40). These receptors play a critical role in hormonalmodulation of gene expression by estrogen and estrogen-like ligands.Signal transduction upon ligand-binding is dependent on characteristicsequence motifs within the receptor protein. These may include a DNAbinding domain (DBD), nuclear localization signals, a ligand-bindingdomain (LBD) and transactivation domains, TAF-1 and TAF-2 which activatetranscription of target genes in a ligand independent or dependentfashion respectively. Estrogen receptors dimerize upon ligand activationand this process precipitates DNA binding (Cowley et al. J Biol Chem1997 Aug. 8; 272(32):19858-62, Pettersson et al. Mol. Endocrinol. 1997;September 11 (10): 1486-96, Pace et al. J. Biol. Chem. 1997;272(41):25832-8). Ligand-bound receptor recognizes specific estrogenresponse elements within the promoter regions of estrogen-regulatedgenes to induce the transactivation response.

It has been shown that members of the estrogen receptor (ER) superfamilyhave multiple subtypes and isoforms. ER-like mRNAs distinct from thewild-type ER mRNA have been identified in many known ER positive tissuesand cell lines. These isoforms have been mostly derived from alternativesplicing of ER mRNAs (Lu et al. Mol Cell Endocrinol. 1998 Mar. 16; 138(1-2):199-203, (Murphy et al., J. Steroid Biochem. Mol. Bio. 1998;65:175-180, Lu et al., Mol. Cell. Endrocrinol. 1998; 16: 199-203, Murphyet al., J. Steroid Biochem. Mol. Bio. 1997; 62: 363-372). Furthermore,there has been strong evidence that some variant/mutant ER mRNAs arestably translated in vivo and that they may have functional role(s)possibly in ER signal transduction (Fuqua et al., Cancer Research, 1992;52, 483-486, Fuqua et al., Cancer Research, 1991; 51, 105-109). Recentlyseveral isoforms of the human ERβ gene have been described (WO 99/07847,Kastner et al. Proc. Natl. Acad. Sci. USA, 1997; 87, 2700-2704, Leroy etal. EMBO J. 10, 59-69, 1991, Zelent et al. EMBO J. 10, 71-81, 1991).Accordingly, these variant ER mRNAs produce novel proteins which differstructurally and exhibit altered physiological functions. For example,some of ER variant receptors possess anomalous transcriptional activitythat may inhibit or enhance the effects of the wild-type receptors. Inaddition, some may act as dominant negative receptors. In one example,adenoviral delivery of a dominant negative ER to ER-positive breastcancer cells effectively suppressed estrogen-stimulated cellproliferation and the hormonal induction of endogenous genes (Lazennecet al. Mol. Endocrinol. 1999; 13(6):969-80.). In another example, fivetranscripts arise from the human estrogen receptor β (ERβ) (Moore et al.Biochem Biophys Res Commun 1998; 247(1):75-8). In this case, full-lengthvariants showed reduced affinity for estrogens and were able to formDNA-binding homodimers and heterodimers with each other and with the ERβsubtype. In another example, the sequence of a splice variant receptor,named ERβcx for c-terminal exchange, diverged at exon 7 (Ogawa et al.Nucleic Acids Res. 1998; 26(15):3505-12). In this case, 61 amino acidsof wild-type protein sequence were substituted with a unique sequenceencoding 26 amino acids. In transcription assays, the relativeexpression of this novel receptor had profound effects onestrogen-induced trans-activation. The evidence suggested that theseeffects were mediated through a heterodimerization mechanism. Thisisoform was shown to preferentially form heterodimers with ERα and wasdemonstrated to behave in a dominant negative fashion against ERαtransactivation in vitro in cotransfection studies. ERβcx was showntherefore to be a potential inhibitor of estrogen (E2) action throughspecific interaction with the ERα isoform (Nucleic Acids Res. 1998;26(15):3505-12).

In summary, ongoing research suggests that there exists complexity inestrogen signaling pathways and ER variants may contribute to estrogenpharmacology differently.

In order to understand the mechanism of estrogen action and ERβregulation of gene transcription, it is important to isolate andcharacterize novel subtypes, variants, and/or isoforms of the ER. Oncethe underlying ER subtype, variant or isoform responsible for aparticular disease state or pathological condition is determined, onemay have a more accurate means of prognostigating the estrogen receptorrelated disease outcome; one may use the presence or amount ofexpression of the novel polynucleotide of the present invention and/orthe polypeptide encoded by such polynucleotide for diagnosing associatedpathological conditions or a susceptibility to an associatedpathological condition; one may accurately follow therapies, developgene specific and isoform specific therapies influenced by ER, and/ormay develop new pharmaceutical drug targets.

Thus, there exists a need to identify new variants and isoforms andtheir protein products for the therapeutic treatment of human diseases.The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid fragmentencoding an estrogen receptor variant polypeptide selected from thegroup consisting of: (a) an isolated nucleic acid fragment encoding SEQID NO:1; (b) an isolated nucleic acid fragment encoding an amino acidsequence having at least 95% identity with the SEQ ID NO:1; (c) anisolated nucleic acid molecule that hybridizes with the isolated nucleicacid fragment of (a) under hybridization conditions of 6×SSC (1M NaCl),45 to 50% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55to 60° C., (d) an isolated nucleic acid fragment that is complementaryto (a), (b) or (c).

In an alternate embodiment, the present invention provides polypeptidesencoded by the nucleotide sequences described above. It is preferredthat the polypeptide of the claimed invention is involved in estrogensignaling pathway.

The invention further provides chimeric constructs comprising theisolated nucleic acid fragment of present invention operatively linkedto suitable regulatory sequences.

Additionally, the invention provides a host cell comprising a chimericconstruct of the present invention or an isolated nucleic acid fragmentof the present invention.

In another embodiment, the invention provides for a vector comprisingthe isolated nucleic acid fragment of the present invention.

Additionally, the present invention provides a host cell comprising avector of the present invention.

In an alternate embodiment, the present invention provides an isolatednucleic acid fragment selected from the group consisting of: (a) anisolated nucleic acid fragment encoding SEQ ID NO:11; an isolatednucleic acid molecule that hybridizes with the isolated nucleic acidfragment of (a) under hybridization conditions of 6×SSC (1M NaCl), 45 to50% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to60° C.; and (c) an isolated nucleic acid fragment that is complementaryto (a), or (b).

In another embodiment, the present invention provides a method forobtaining a nucleic acid fragment encoding the polypeptide of presentinvention, the method comprising: (a) probing a genomic library with allor a portion of a nucleic acid fragment as set forth in SEQ ID NO:2; (b)identifying a DNA clone that hybridizes with the nucleic acid fragmentof step (a); and (c) determining the sequence of the nucleic acidfragment that comprises the DNA clone identified in step (b).

In another embodiment, the present invention provides a method ofdetecting the presence of a nucleic acid fragment of present inventionin a sample, the method comprising: (a) contacting said sample with theoligonucleotide of claim 29; and (b) determining whether theoligonucleotide detects the nucleic acid fragment.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 depicts inverse PCR strategy for cloning novel ERβ 3′ flankingsequences from human cDNA library (referred to in Example 1).

FIG. 2 shows the nucleotide sequence and predicted amino acidcomposition of ERβcx2 clone isolated from a fetal brain cDNA library(referred to in Example 2).

FIG. 3 shows comparison of selected human ERβ variants (referred to inExample 3).

FIG. 4 shows genomic organization of ERβcx2 (referred to in Example 4).

FIG. 5 shows expression of ERβcx2 transcripts in human tissues (referredto in Example 5).

FIG. 6 shows relative abundance of ERα, ERβ and ERβcx2 variants in humantestis RNA by RT-PCR analysis (referred to in Example 6).

FIG. 7 shows results of transactivation of ERE-tk-Luciferase reporter byestrogen receptor variants, ERα, ERβ and ERβcx2 (referred to in Example7).

FIG. 8 shows immunoblot analysis of ERα, ERβ and ERβcx2 (referred to inExample 7).

FIG. 9 shows dominant activity of ERβcx2 in the ERα signaling pathway(referred to in Example 7).

FIG. 10 shows dominant activity of ERβcx2 in the ERβ signaling pathway(referred to in Example 7).

The following 23 sequence descriptions and sequence listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. (“Requirements for Patent Applications containingnucleotide sequences and/or Amino Acid Sequence Disclosure—the SequenceRules”) and consistent with World Intellectual Property Organization(WIPO) Standard ST.25 (1998) and the sequence listing requirements ofthe EPO and PCT (Rules 5.2 and 4.95(a-bis) and Section 208 and Annex Cof the Administrative Instructions). The Sequence Descriptions containsthe one letter code for nucleotide sequence characters and the threeletter codes for amino acids as defined in conformity with theIUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030(1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which areherein incorporated by reference. The symbols and format used fornucleotide and amino acid sequence data comply with the rules set forthin 37 C.F.R. §1.822. The present invention utilizes Lasergene software,Version 4.5, DNASTAR, Madison, Wis.

SEQ ID NO:1 is the deduced amino acid sequence of ERβcx2.

SEQ ID NO:2 is the nucleotide sequence that codes for ERβcx2.

SEQ ID NO:3 is the amino acid sequence coded by ERβcx2 exon 7.

SEQ ID NO:4 is the nucleotide sequence ERβcx2 exon 7 and 3′ UTR.

SEQ ID NO:5 is the nucleotide sequence of the primer, ERβ GSP2.

SEQ ID NO:6 is the nucleotide sequence of the primer, ERβ GSP3.

SEQ ID NO:7 is the nucleotide sequence of the primer, ERβ GSP6.

SEQ ID NO:8 is the nucleotide sequence of the vector primer, T7 Sport.

SEQ ID NO:9 is the nucleotide sequence of the vector primer,Sport-forward.

SEQ ID NO:10 is the nucleotide sequence of the forward primer, GSP start3.

SEQ ID NO:11 is the nucleotide sequence of the reverse primer, ERβ x-3.

SEQ ID NO:12 is the nucleotide sequence of the primer, GSP7-forward.

SEQ ID NO:13 is the nucleotide sequence of the primer, GSP7-reverse.

SEQ ID NO:14 is the nucleotide sequence of the primer, ERβ x-1.

SEQ ID NO:15 is the nucleotide sequence of the primer, ERβ x-2.

SEQ ID NO:16 is the nucleotide sequence of the primer, P2.

SEQ ID NO:17 is the nucleotide sequence of the primer, P3.

SEQ ID NO:18 is the nucleotide sequence of the primer, hERα-5′.

SEQ ID NO:19 is the nucleotide sequence of the primer, hERα-3′.

SEQ ID NO:20 is the nucleotide sequence of the primer, hERβ-5′.

SEQ ID NO:21 is the nucleotide sequence of the primer, hERβ-3′.

SEQ ID NO:22 is the nucleotide sequence of the primer, hERβcx2-5′.

SEQ ID NO:23 is the nucleotide sequence of the primer, hERβcx2-3′.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have succeeded in identifying and characterizing a geneencoding for a novel estrogen receptor beta variant (ERβcx2). This newlyidentified gene produces a variant that differs structurally andfunctionally from the known estrogen receptor proteins. This novel humanvariant differs in its C-terminal sequence from the wild-type estrogenreceptor in that it's C-terminal 61 amino acids are replaced by a uniquesequence of 7 amino acids. Applicants have also provided evidence thatthis novel receptor is functional in that it interacts with the estrogenreceptor and that it may act as a dominant negative mutant withinhibitory effects on the ER (e.g. ERα, ERβ) signaling pathway. TheApplicants have also analyzed tissue expression of this novel variantand showed that it is most abundant in Testis.

The following definitions are provided for the full understanding ofterms and abbreviations used in this specification.

The abbreviations in the specification correspond to units of measure,techniques, properties or compounds as follows: “min” means minutes, “h”means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM”means millimolar, “M” means molar, “mmole” means millimole(s), “kb”means kilobase, “bp” means base pair(s), and “IU” means InternationalUnits.

“Polymerase chain reaction” is abbreviated PCR.

“Reverse transcriptase polymerase chain reaction” is abbreviated RT-PCR.

“Estrogen receptor” is abbreviated ER.

“DNA binding domain” is abbreviated DBD.

“Ligand binding domain” is abbreviated LBD.

“Untranslated region” is abbreviated UTR.

“Sodium dodecyl sulfate” is abbreviated SDS.

In the context of this disclosure, a number of terms shall be utilized.As used herein, the term “nucleic acid molecule” refers to the phosphateester form of ribonucleotides (RNA molecules) or deoxyribonucleotides(DNA molecules), or any phosphoester analogs, in either single strandedform, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA andRNA-RNA helices are possible. The term nucleic acid molecule, and inparticular DNA or RNA molecule, refers only to the primary and secondarystructure of the molecule, and does not limit it to any particulartertiary forms. Thus, this term includes double-stranded DNA found,inter alia, in linear (e.g., restriction fragments) or circular DNAmolecules, plasmids, and chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of DNA (i.e., the strandhaving a sequence homologous to the mRNA). A “recombinant DNA molecule”is a DNA molecule that has undergone a molecular biologicalmanipulation.

The terms “polynucleotide” or “nucleotide sequence” is a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA, and meansany chain of two or more nucleotides. A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double orsingle stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and antisense polynucleotide.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases, for examplethio-uracil, thio-guanine and fluoro-uracil.

The polynucleotides may be flanked by natural regulatory (expressioncontrol) sequences, or may be associated with heterologous sequences,including promoters, internal ribosome entry sites (IRES) and otherribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The terms “nucleic acid” or “nucleic acid sequence” or “polynucleotide”may be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

Variant(s) refer to polypeptides that differ from a referencepolypeptide respectively. Generally, the differences between thepolypeptide that differs in amino acid sequence from referencepolypeptide, and the reference polypeptide are limited so that the aminoacid sequences of the reference and the variant are closely similaroverall and, in some regions, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, deletions, additions, fusions and truncations, which maybe present in any combination. Additionally, a variant may be a fragmentof a polypeptide of the invention that differs from a referencepolypeptide sequence by being shorter than the reference sequence, suchas by a terminal or internal deletion. A variant of a polypeptide of theinvention also includes a polypeptide which retains essentially the samebiological function or activity as such polypeptide e.g., precursorproteins which can be activated by cleavage of the precursor portion toproduce an active mature polypeptide. Moreover, a variant may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a precursor protein sequence. A variant of thepolypeptide may also be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Among polypeptide variants in this regard arevariants that differ from the aforementioned polypeptides by amino acidsubstitutions, deletions or additions. The substitutions, deletions oradditions may involve one or more amino acids. Alterations in thesequence of the amino acids may be conservative or non-conservativeamino acid substitutions, deletions or additions. All such variantsdefined above are deemed to be within the scope of those skilled in theart from the teachings herein and from the art.

The ERβ variant described herein that is designated ERβcx2 (SEQ IDNO:1), is homologous to the previously known ERβ and the two genes havecommon N-terminal amino acid sequences corresponding to the amino acidsencoded by exons 1-6 of the ERβ gene and the two genes differ in theirC-terminal in that the C-terminal exon 7 sequence of the wild-type ERβis replaced by a unique sequence of 7 amino acids. By virtue of thepartial identity and partial divergence of their amino acid sequences,the variant and the known homologous may have some functionality incommon but may differ in other functions. For example, wild-type ERβ isknown to be a weak transcriptional activator where as ERβcx is dominantnegative receptor in that it is transcriptionally inactive and that maydimerize with the ER and inactivate biological functions of thewild-type receptor.

“Dominant negative variant” refers to a variant that can act in adominant negative fashion. Dominant negative usually is a result of amutation creating a negative phenotype which is dominant when expressedin the presence of the wild-type protein or background. Such alterationsof the nucleotide sequence encoding the ligand binding domain includebut are not limited to deletions or substitutions of critical amino acidresidues within the domains that are required for ligand binding. Forexample, dominant negative variant forms of ER (estrogen receptor) maybe a result of modification of the nucleotide sequence of the ligandbinding domain of the wild-type gene which would eliminate ligandbinding ability of the wild-type receptor. These variants or alteredforms of the estrogen receptors may be transcriptionally inactive andmay suppress the biological functions of the wild-type ER, potentiallyby heterodimerizing with the wild-type ER.

“Splice variant” refers to cDNA molecules produced from RNA moleculesinitially transcribed from the same genomic DNA sequence but which haveundergone alternative RNA splicing. Alternative RNA splicing occurs whena primary RNA transcript undergoes splicing, generally for the removalof introns, which results in the production of more than one mRNAmolecule each of them may encode different amino acid sequences. Theterm splice variant may also refer to the proteins encoded by the abovecDNA molecules. The splice variant may be partially identical insequence to the known homologous gene product.

“Branch site” and “3′ acceptor sites” refer to consensus sequences of 3splice junctions in eukaryotic mRNAs. Almost all introns begin with GUand end with AG (. From the analysis of many exon-intron boundaries,extended consensus sequences of preferred nucleotides at the 5 and 3ends have been established. In addition to AG, other nucleotides justupstream of the 3 splice junction also are important for precisesplicing (i.e., branch site consensus, YNYURAY and 3′ acceptor site,(Y)nNAG G).

The term “polynucleotide encoding polypeptide” encompasses apolynucleotide which may include only the coding sequence as well as apolynucleotide which may include additional coding or non-codingsequence.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (Sambrook, J. et al. eds., Molecular Cloning: ALaboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY.Vols. 1-3 (ISBN 0-87969-309-6). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Forpreliminary screening for homologous nucleic acids, low stringencyhybridization conditions, corresponding to a T_(m) of 55°, can be used,e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide,5×SSC, 0.5% SDS). Moderate stringency hybridization conditionscorrespond to a higher T_(m), e.g., 40% formamide, with 5× or 6×SCC.High stringency hybridization conditions correspond to the highestT_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requires that thetwo nucleic acids contain complementary sequences, although depending onthe stringency of the hybridization, mismatches between bases arepossible. The appropriate stringency for hybridizing nucleic acidsdepends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (Sambrook et al. eds.,Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring HarborLaboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6), 9.50-9.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (Sambrook et al. eds.,Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring HarborLaboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6), 11.7-11.8).

The present invention particularly contemplates nucleic acid sequencesthat hybridize under stringent conditions to the ERβcx2 variant codingsequences described herein and complementary sequences thereof. For thepurposes of this invention, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the nucleic acid sequences. Accordingly, thepresent invention also includes isolated nucleic fragments that arecomplementary to the complete sequences as reported in the accompanyingSequence Listing as well as those that are at least 95% identical tosuch sequences, and polynucleotides having sequences that arecomplementary to the aforementioned polynucleotides. The polynucleotidesof the present invention that hybridize to the complement of ERβcx2variant coding sequences described herein preferably encode polypeptidesthat retain substantially the same biological function or activity asthe mature ERβcx2 polypeptide encoded by the cDNA of SEQ ID NO:2.

A “substantial portion” of an amino acid or nucleotide sequencecomprising enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computerautomated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLASTO. In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The present specification teaches partial or completeamino acid and nucleotide sequences encoding one or more particular ERvariants. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the present invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

“Identity” or “similarity”, as known in the art, are relationshipsbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated by known methods such as thosedescribed in: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991. Methods commonly employed to determine identity or similaritybetween sequences include, but are not limited to those disclosed inCarillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988).Methods to determine identity and similarity are codified in publiclyavailable computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F.et al., J Molec. Biol. 215: 403 (1990)).

“Homologous” refers to the degree of sequence similarity between twopolymers (i.e. polypeptide molecules or nucleic acid molecules). Thehomology percentage figures referred to herein reflect the maximalhomology possible between the two polymers, i.e., the percent homologywhen the two polymers are so aligned as to have the greatest number ofmatched (homologous) positions.

The term “percent homology” refers to the extent of amino acid sequenceidentity between polypeptides. The homology between any two polypeptidesis a direct function of the total number of matching amino acids at agiven position in either sequence, e.g., if half of the total number ofamino acids in either of the sequences are the same then the twosequences are said to exhibit 50% homology.

The term “fragment”, “analog”, and “derivative” when referring to thepolypeptide of the present invention (SEQ ID NO:1), refers to apolypeptide which may retain essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a precursorprotein which can be activated by cleavage of the precursor proteinportion to produce an active mature polypeptide. The fragment, analog,or derivative of the polypeptide of the present invention (SEQ ID NO:1)may be one in which one or more of the amino acids are substituted witha conserved or non-conserved amino acid residue and such amino acidresidue may or may not be one encoded by the genetic code, or one inwhich one or more of the amino acid residues includes a substituentgroup, or one in which the polypeptide is fused with a compound such aspolyethylene glycol to increase the half life of the polypeptide, or onein which additional amino acids are fused to the polypeptide such as asignal peptide or a sequence such as polyhistidine tag which is employedfor the purification of the polypeptide or the precursor protein. Suchfragments, analogs, or derivatives are deemed to be within the scope ofthe present invention.

The polypeptide and the polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalor native environment (e.g., the natural environment if it is naturallyoccurring). Therefore, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated by human intervention from someor all of the coexisting materials in the natural system, is isolated.For example, an “isolated nucleic acid fragment” is a polymer of RNA orDNA that is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of the environment in which it isfound in nature. Similarly, the term “substantially purified” refers toa substance, which has been separated or otherwise removed, throughhuman intervention, from the immediate chemical environment in which itoccurs in Nature. Substantially purified polypeptides or nucleic acidsmay be obtained or produced by any of a number of techniques andprocedures generally known in the field.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the polynucleotide sequence without effecting the aminoacid sequence of an encoded polypeptide. Accordingly, the presentinvention relates to any nucleic acid fragment that encodes all or asubstantial portion of the amino acid sequence encoding the instantERβcx2 protein as set forth in SEQ ID NO:1. The skilled artisan is wellaware of the “codon-bias” exhibited by a specific host cell to usenucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well known procedures, orautomated chemical synthesis can be performed using one of a number ofcommercially available machines. Accordingly, the genes can be tailoredfor optimal gene expression based on optimization of nucleotide sequenceto reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determiningpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ noncodingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” or “chimeric construct” refers toany gene or a construct, not a native gene, comprising regulatory andcoding sequences that are not found together in nature. Accordingly, achimeric gene or chimeric construct may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature.“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign” gene refers to a gene not normallyfound in the host organism, but which is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Gene control sequence” refers to the DNA sequences required to initiategene transcription plus those required to regulate the rate at whichinitiation occurs. Thus a gene control sequence may consist of thepromoter, where the general transcription factors and the polymeraseassemble, plus all the regulatory sequences to which gene regulatoryproteins bind to control the rate of these assembly processes at thepromoter. For example, the control sequences that are suitable forprokaryotes may include a promoter, optionally an operator sequence, anda ribosome binding site. Eukaryotic cells are known to utilizepromoters, enhancers, and/or polyadenylation signals.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency.

The term “operatively linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperatively linked with a coding sequence when it is capable ofaffecting the expression of that coding sequence (i.e., that the codingsequence is under the transcriptional control of the promoter). Codingsequences can be operatively linked to regulatory sequences in sense orantisense orientation.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein.

“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms. Over expression of the polypeptideof the present invention may be accomplished by first constructing achimeric gene or chimeric construct in which the coding region isoperatively linked to a promoter capable of directing expression of agene or construct in the desired tissues at the desired stage ofdevelopment. For reasons of convenience, the chimeric gene or chimericconstruct may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene or chimeric construct may also comprise one or moreintrons in order to facilitate gene expression. Plasmid vectorscomprising the instant chimeric gene or chimeric construct can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host cells. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene or chimeric construct. The skilled artisanwill also recognize that different independent transformation eventswill result in different levels and patterns of expression (Jones et al.(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics218:78-86), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

A “cassette” refers to a DNA coding sequence or segment of DNA thatcodes for an expression product that can be inserted into a vector atdefined restriction sites. The cassette restriction sites are designedto ensure insertion of the cassette in the proper reading frame.Generally, foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell alongwith the transmissible vector DNA. A segment or sequence of DNA havinginserted or added DNA, such as an expression vector, can also be calleda “DNA construct.”

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides include but are not limited tointracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

“Clone” refers to a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” refers to a clone of a primarycell that is capable of stable growth in vitro for several generations.

The present invention incorporates by reference methods and techniqueswell known in the field of molecular and cellular biology. Thesetechniques include, but are not limited to techniques described in thefollowing publications: Old, R. W. & S. B. Primrose, Principles of GeneManipulation: An Introduction To Genetic Engineering (3d Ed. 1985)Blackwell Scientific Publications, Boston. Studies in Microbiology;V.2:409 pp. (ISBN 0-632-01318-4), Sambrook, J. et al. eds., MolecularCloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor LaboratoryPress, NY. Vols. 1-3. (ISBN 0-87969-309-6), Miller, J. H. & M. P. Caloseds., Gene Transfer Vectors For Mammalian Cells (1987) Cold SpringHarbor Laboratory Press, NY. 169 pp. (ISBN 0-87969-198-0). The DNAcoding for the protein of the present invention may be any one providedthat it comprises the nucleotide sequence coding for the above-mentionedprotein of the present invention.

Accordingly, the present invention relates to the vectors that includepolynucleotides of the present invention, host cells that geneticallyengineered with vectors of the present invention such as cloning vectoror expression vector and to the production of polypeptides of thepresent invention by recombinant techniques.

The present invention further relates to a method of production of thepolypeptide of the present invention by expressing a polynucleotideencoding the polypeptide of the present invention in a suitable host andrecovering the expressed product employing known recombinant techniques.The polypeptide of the present invention can also be synthesized bypeptide synthesizers. Host cells can be engineered with the vectors ofthe present invention. The host organism (recombinant host cell) may beany eukaryotic or prokaryotic cell, or multicellular organism. Suitablehost cells include but are not limited to mammalian cells (e.g. such asHuman hepatoma cells (HepG2), Chinese hamster ovary cells (CHO), themonkey COS-1 cell line, the mammalian cell CV-1), amphibian cells (e.g.Xenopus egg cell), yeast cells (Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris), and insect cells.Furthermore, various strains of E. coli (e.g., DH5α, HB101, MC1061) maybe used as host cells in particular for molecular biologicalmanipulation.

The vectors may be cloning vectors or expression vectors such as in theform of a plasmid, a cosmid, or a phage or any other vector that isreplicable and viable in the host cell. The engineered host cells can becultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying thepolynucletide of the present invention. The culture conditions such aspH, temperature, and the like, are those suitable for use with the hostcell selected for expression of the polynucleotide are known to theordinarily skilled in the art.

Plasmids generally are designated herein by a lower case “p” precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. The plasmids herein are either commercially available, publiclyavailable on unrestricted bases, or can be constructed from availableplasmids by routine application of well-known, published procedures.Additionally, many plasmids and other cloning and expression vectorsthat can be used in accordance with the present invention are well knownand readily available to those of skill in the art. Moreover, those ofskill readily may construct any number of other plasmids suitable foruse in the invention. The properties, construction and use of suchplasmids, as well as other vectors, in the present invention will bereadily apparent to those of skill from the present disclosure.

The appropriate DNA sequence may be inserted into the vector by avariety of the procedures known in the art.

The DNA sequence in the expression vector may be operatively linked toan appropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. Such promoters include but are not limited to SV40, humancytomegalovirus (CMV) promoters (e.g. pcDNA 3.1 vector or any form ofthe pcDNA series), SP6, T7, and T3 RNA polymerase promoters. Theexpression vector may also include a ribosome binding site fortranslation initiation, a transcription terminator, and an appropriatesequences for amplifying the expression. The expression vector may alsoinclude one or more selectable marker genes to provide a specificphenotype for the selection of transformed host cells such as neomycinresistance for eukaryotic cells or ampicillin resistance for E. coli.

The gene may be placed under the control of a promoter, ribosome bindingsite (for bacterial expression), suitable gene control sequence, orregulatory sequences so that the DNA sequence encoding the protein istranscribed into RNA in the host cell transformed by a vector containingthis expression construct. Such promoters include but are not limited toSV40, human cytomegalovirus (CMV) promoters (e.g. pcDNA 3.1 vector orany form of the pcDNA series), SP6, T7, and T3 RNA polymarase promoters.In some cases it may be desirable to add sequences which cause thesecretion of the polypeptide from the host cell, with subsequentcleavage of the secretory signal.

It may also be desirable to reduce or eliminate expression of genesencoding the polypeptide of the present invention for some applications.In order to accomplish this, a chimeric gene or a chimeric constructdesigned for co-suppression of the instant polypeptide can beconstructed by linking a gene or gene fragment encoding that polypeptideto a promoter sequences. Alternatively, a chimeric gene or chimericconstruct designed to express antisense RNA for all or part of theinstant nucleic acid fragment can be constructed by linking the gene orgene fragment in reverse orientation to a promoter sequences. Either theco-suppression or antisense chimeric genes could be introduced intodesired host cell via transformation wherein expression of thecorresponding endogenous genes are reduced or eliminated.

The polynucleotide of the present invention, may be in the form of RNAor in the form of DNA, which DNA includes cDNA and synthetic DNA. TheDNA may be single stranded or double stranded. If it is single stranded,it may be the coding strand or non-coding (antisense) strand. The codingsequence may be identical to the coding sequence of SEQ ID NO:2 or maybe a different coding sequence which the coding sequence, as a result ofdegeneracy or redundancy of the genetic code, encodes for the samepolypeptide.

The present invention may include variants of the herein-above describedpolynucleotides which encode fragments, analogs, and derivatives of thepolynucleotides characterized by the deduced amino acid sequence of SEQID NO:1. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

The polynucleotide of the present invention, may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequencecharacterized by the DNA sequence of the SEQ ID NO:2. An Allelic variantis an alternate form of a polynucleotide sequence which which may have asubstitution, deletion, or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The polynucleotide which encodes for the mature polypeptide, i.e.ERβcx2, may include only the coding sequence for the mature polypeptideor the coding sequence for the mature polypeptide and additionalsequence such as gene control sequence, regulatory or secretorysequence.

The present invention therefore includes polynucleotides wherein thecoding sequence for the mature polypeptide may be operatively linked inthe same reading frame to a polynucleotide sequence which aids inexpression and secretion of a polypeptide from a host cell. For example,a signal peptide. The polynucleotide may also encode for a precursorprotein.

The polynucleotide of the present invention may also have the codingsequence fused in frame to a marker sequence, such as hexa-histidine tag(Qiagen Inc.), at either 3′ or 5′ terminus of the gene to allowpurification of the polypeptide of the present invention.

The polypeptide of the present invention may be produced by growingsuitable host cells transformed by expression vector described aboveunder conditions whereby the polypeptide of the interest is expressed.The polypeptide may then be isolated and purified. Methods of thepurification of proteins from cell cultures are known in the art andinclude but not limited to ammonium sulfate precipitation, anion orcation exchange chromatography, and affinity chromatography.

Cell-free translation systems may also be employed to produce thepolypeptides of the present invention using the RNAs derived from thepolynucleotides of the present invention.

Large-scale production of cloned ERβcx2 would enable the screening oflarge numbers of ERβcx2 analogs, and would facilitate the development ofnew or improved agonists and antagonists in the clinical therapy ofestrogen related disorders. More specifically, the screening of largenumbers of analogs for ERβcx2 activity could lead to development ofimproved drugs for use in clinical therapy of cancer, osteoporosis,cardiovascular disorder, etc.

The novel ERβcx2 exon 7 (SEQ ID NO: 4) sequence may be used to generatea dominant negative repressor of estrogen-induced transcription. TheERβcx2 exon 7 (SEQ ID NO: 4) sequence could be incorporated into any oneof the existing variants such as estrogen receptor subtypes α, β, and/orvarious isoforms which are generated by alternative splicing. Theresulting new polypeptides comprising the amino acid sequence encoded bySEQ ID NO: 4 which is set forth in SEQ ID NO:3 may generate a dominantnegative repressor of estrogen-induced transcription.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences added and/orwith targeting sequences that are already present removed. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of utility may be discovered inthe future.

Furthermore, the polypeptides of the present invention or cellsexpressing them may be used as immunogen to prepare antibodies bymethods known to those skilled in the art. For example, thesepolypeptides encoded by SEQ ID NO: 2 or any portion of SEQ ID NO: 2and/or encoded by SEQ ID NO:4 or cells expressing any of theaforementioned polypeptides may be used as immunogens. These antibodiescan be polyclonal or monoclonal and may include chimeric, single chain,and Fab fragments or the products of the Fab expression library. Theantibodies are useful for detecting the polypeptide of the presentinvention in situ in cells or in vitro in cell extracts.

In addition, the polypeptide of the present invention can be used astargets to facilitate design and/or identification of compounds that maybe useful as drugs. In particular, these compounds may be used to treatdiseases resulting from alterations in estrogen signaling pathways suchas cancer, osteoporosis, cardiovascular diseases. In addition, thepolypeptides of the present invention may be used to identify additionaltargets (e.g. co-activator or co-repressor proteins) that may influenceestrogen signaling. Various uses of the ER variant of the presentinvention include but are not limited to therapeutic modulation ofpathophysiologic estrogen signaling (e.g. gene delivery approaches, genesilencing approaches, protein therapeutics antibody therapeutics),diagnostic utility, pharmaceutical drug targets, identification ofreceptor-based agonists or antagonists, and study of the molecularmechanisms of estrogen action.

Accordingly, dominant negative ER variants offer utility for therapeuticmodulation of pathophysiologic estrogen signaling. Estrogen-inducedsignaling mediated by estrogen receptors is affected by aberrant ERs.Variants may produce a constituitively active phenotype contributing tocarcinogenesis or act as dominant negative modulators resulting in lossof estrogen responsiveness and rendering antiestrogen therapyineffective. The delivery of transcriptionally altered ERs termeddominant negative mutants for example to breast cancer cells holdspromise as a strategy to treat breast cancer. Furthermore, smallmolecules may be developed to efficiently deliver dominant negative ERsto mammalian cells circumventing the current inefficient deliveryapproaches and those requiring the use of recombinant virus (Hussey etal. Organic Letters 2002; 4: 4145-418). This therapeutic approach wouldserve as a viable alternative to the use of antiestrogens or potentcompetitive antagonists of estrogen receptors such as tamoxifan,particularly since most breast cancers eventually become resistant tothese types of antihormone therapeutics.

Moreover, in cells refractory to antiestrogen therapy due to phenotypicexpression of endogenous dominant negative ER variant of the presentinvention, gene-silencing approaches such as antisense, siRNA (smallinterfering RNA), etc might be employed as strategies to induce orstimulate estrogen signaling. Additionally, the novel variant of thepresent invention may be used to make fusion ER variants which may beemployed toward the development of receptor-based agonists andantagonists.

The present invention relates to a novel protein, characterized in thatit comprises the amino acid sequence given in SEQ ID NO:1.

In one embodiment, the invention provides polynucleotides (DNA or RNA)which encodes such a polypeptide. The nucleotide sequence of cloned PCRproducts were compared with existing Genbank entries using the BLASTsearch program. An EST clone (Genbank #AA829530) containing a largerfragment of the novel exon 7 sequence was identified. This clone ofapproximately 2.0 Kb was obtained (IMAGE Consortium) and sequenced.Splice acceptor consensus sites were identified using the GCG/SeqWebprogram FindPatterns. In addition, the present invention providesfeatures such as a polyadenylation signal and poly A tail as evident inthe 3′ UTR following the stop codon (FIG. 2). Nucleotide boundariesconsistent with a consensus 5′ splicing junction sequence also implythat the variant protein may be generated by alternative splicing.

In particular the invention provides a polynucleotide encoding a humanERβ variant and characterized in that it comprises the DNA sequencegiven in SEQ ID NO:2. The polynucleotide having the DNA sequence givenin SEQ ID NO:2 was obtained from a fetal brain by conventionaltechniques. For example, a cDNA fragment encoding a portion of the novelER variant was initially isolated from a fetal brain cDNA library usingan inverse PCR approach (FIG. 1). Sequence analysis revealed thepolypeptide as a novel human variant (FIG. 2). The novel sequencediverges at the wild-type ERβ exon 6-7 junction where the C-terminal 61amino acids of wild-type ERβ are replaced in the variant by a uniquesequence encoding 7 amino acids prior to termination as indicated.

The invention also provides a novel oligonucleotide as set forth in SEQID No: 11 (ERβ x-3) which spans exon 6 of the wild-type ERβ and thenovel exon 7 boundary. The novel primer can act as PCR primer in theprocess herein described to determine whether or not the ERβcx2 geneidentified whole or in part are transcribed in various tissues. Thenovel primer of the present invention may be used in combination withvarious ER primers (e.g. ERβ wild-type or ERβcx nucleotide sequence) todetect the presence of the novel variant of the present invention. It isrecognized that such sequence will have utility in diagnosis of varioushealth states which may be directly or indirectly related to thepresence of this mutant from of estrogen receptor.

In another embodiment, the sequence of the PCR amplified productupstream of the exon 6-7 junction was analyzed and showed almost 100%nucleotide sequence similarity with ERβ (FIG. 3). The ERβcx2 cDNA wasconfirmed using PCR amplification and the nucleotide sequence up to endof exon 6 is invariant with the wild-type ERβ receptor. One conservativenucleotide change was observed in the N-terminus of the amplicon,potentially a polymorphism. Adenine (A) is substituted for guanine (G)at nucleotide 35 (4th nucleotide relative to the initiation codon ATG).In the translated sequence this nucleotide change results in an aminoacid substitution of aspartic acid with asparagine relative to thepublished ERβ sequence (D→N) or (ASP→ASN). Comparison with ERβ showssequence identity with the A/B domain located in the N-terminuscontaining a transactivation function (AF-1), the DNA Binding Domain(DBD), and the hinge region. This homology however, extends to only aportion of the critical ligand binding domain (LBD), involved in bindingligand, dimerization, and transactivation.

In another embodiment, genomic organization analysis of the novelsequence confirmed that the novel ERβcx2 sequence resides about 3.4 kbdownstream of ERβ exon 7 and its 3′ UTR (FIG. 4). The genomic locationof the novel exon 7 sequences was defined using two PAC genomic clonesisolated for this purpose. Intronic consensus splicing motifs wereidentified upstream of the 7 amino acid coding sequence. Consensussplice signals for a branch site and 3′ acceptor site, were foundimmediately upstream of the alternate exon 7 sequence. Therefore, theexchange of the last exon of ERβ may have been occurred by analternative splice mechanism.

In yet another embodiment, RT-PCR analysis revealed that the variantmRNA transcript of the present invention is most abundant in Testis.However, with increasing amplification cycles, expression was observedin ovary, small intestine, spleen, and thymus (FIG. 5).

In yet another embodiment, the relative expression of ERα, ERβ, and thenovel ERβcx2 variants was determined in human testis RNA by semiquantitative RT-PCR analysis using primers specific for ERα, ERβ, andERβcx2. The data suggest that transcripts for the novel variant arepresent at a comparable level to the wild-type receptors in total RNAfrom testis (FIG. 6). Furthermore, the expression of ERβcx2 mRNA roughlyparalleled that observed for ERβcx mRNA identified in testis, ovary,prostate and thymus (Ogawa et al. Nucleic Acids Res. 1998;26(15):3505-12). However, ERβ is predominant in thymus, testis, ovary,and spleen (Mosselman et al. FEBS Lett 1996 Aug. 19; 392(1):49-53).

In another embodiment, functional studies were performed to define thetransactivation properties of the ERβcx2 protein (FIG. 7). Substitutionof exon 7 sequences disrupts the LBD inactivating the receptortransactivation function. In transient transfection experiments, ERβcx2was unable to induce transcription from ERE-Luciferase reporterconstructs coexpressed in HepG2 cells. Immunoblot analysis of proteinexpression confirmed that the variant isoform, ERβcx2, was synthesizedin the transfection experiments, suggesting it is nonfunctional (i.e.,cannot bind ligand or transactivate) (FIG. 8).

In yet another embodiment, cotransfection experiments were performed andidentified the ERβcx2 variant as a possible dominant negative mutantwith inhibitory effects on the ERβ signaling pathway (ERα signaling mayalso be affected) (FIGS. 9 and 10).

As a naturally occurring dominant negative mutant, the novel ERβcx2 maybe used for targeting of specific receptor interactions as a distinctapproach in identification of tissue selective estrogen agonists andantagonists.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Cloning of Human ERβ Variant

A human fetal brain cDNA library constructed in pCMV-Sport 4.0/2.2vector (Invitrogen, Carlsbad, Calif.), was screened for novel ER,isoforms using a modification of an inverse PCR protocol. The primaryround of inverse PCR amplification utilized two gene specific primers:ERβ GSP2: GCTCAGCCTGTTCGACCAAGTGCGG (forward) (SEQ ID NO:5), and ERβGSP3: CACAAAGCCGGGAATCTTCTTGGCC (reverse) (SEQ ID NO:6). Reactionconditions were those recommended by the manufacturer of the Expand™Long Template PCR System (BMB, Indianapolis, Ind.).

A second round of PCR was performed using 1 ml of this reaction mixturein the secondary reaction containing one nested gene-specific primer,ERβ GSP6, GAAGCTGGCTCACTTGCTGAACGC (forward) (SEQ ID NO:7) and one ofthe vector primers, T7-Sport: TAATACGACTCACTATAGGGAGAGA (SEQ ID NO:8) orSport-forward: TATGACGTCGCATGCACGCGTAAG (SEQ ID NO:9) with an annealingtemperature of 60° C. for 40 cycles. The resultant products were gelpurified and subcloned into pCR2.1 (Invitrogen, Carlsbad, Calif.) forsequence analysis using the ABI 3700 DNA Analyzer (Applied Biosystems,Foster City, Calif.). This clone was named pERβcx2.

Example 2 Sequencing and Preliminary Analysis of the Clone

The sequence of the clone was determined as described in Example 1. Thenucleotide sequence and predicted amino acid composition of pERβcx2 isshown in FIG. 2. This sequence represents one of three identical clonesisolated from a fetal brain cDNA library. The N-terminal portion of thenucleotide sequence corresponds to exon 6 of the human ERβ cDNA,Genebank Accession # AB006590, AF051427, and X99101. The novel sequencediverges however at the wild-type ERβ exon 6-7 junction where thec-terminal 61 amino acids of wild-type ERβ are replaced in the variantby a unique sequence encoding 7 amino acids prior to termination asindicated.

Example 3 Isolation and Characterization of a Full-Length cDNA Clone ofNovel ERβ Isoform

A full-length cDNA ERβ was isolated from testis total RNA (BDBiosciences Clontech, Palo Alto, Calif.) by RT-PCR (Pfu polymerase). Theforward primer was designed to initiate directly upstream of the firstATG and the reverse primer to recognize the junction of exon 6 and thenovel exon 7 sequence as indicated in FIG. 3. The forward primer, GSPstart 3, CAAGGTGTTTTCTCAGCTGTTATCTCAAGACATGG (SEQ ID NO:10) initiatesupstream of the first ATG of the full-length ERβ cDNA, and the reverseprimer CCAAATGTAAAGCCTCGCATGCCTGA (SEQ ID NO:11) (ERβ x-3) spans theexon 6 and unique exon 7 boundary. Two successive rounds of PCRamplification (40 cycles each) were performed with 5 μl of the firstreaction mixture transferred to the second reaction. The finalamplification product was cloned into bluescript SK (Stratagene, JaJolla, Calif.) and sequenced (FIG. 3).

Example 4 Genomic Organization

Sequence information derived from human PAC clones was used to determinethe physical genomic location of the c-terminal ERβcx2 sequencesrelative to the human ERβ gene. Human PAC genomic clones (GenomeSystems,) were identified using primers specific for exon 7 sequences inthe ERβ gene (GSP7-forward ACAAGGGCATGGAACATCTGCTCAAC (SEQ ID NO:12) andGSP7-reverse CTGAGACTGTGGGTTCTGGGAGCC (SEQ ID NO:13)). These clones wererescreened with primers specific for the novel exon 7 sequence usingprimers, ERβ x-1 GCTTTACATTTGGGCCTTGTAGA (SEQ ID NO:14) and ERβ x-2AACTCTCTGCGACAGTGCCATAGA (SEQ ID NO:15).

The DNA isolated from the PAC clones was further characterized using aPCR strategy and sequence information derived from EST clone #AA829530.The GSP7 forward primer (P1 in FIG. 4), recognizing the wild-type ERβexon 7 sequence was combined with reverse primers designed to amplifyEST #AA829530 sequences. Reverse oligomers; P2(CGAGGTCTTACTAGCAAAAACCAGTCTTGG) (SEQ ID NO:16) and P3(CAGAGCAGCAAACATTCATTTCTACAAGG) (SEQ ID NO:17) recognize the 5′ and 3′ends of the EST sequence, respectively. Two PCR products were generatedwhich differed in size by approximately 2 Kb; the size of the EST. Thesmaller product was cloned into bluescript SK (Stratagene, La Jolla,Calif.) and sequenced.

Example 5 Tissue Distribution

Tissue distribution studies were performed using PCR and Multiple TissuecDNA panels from (MTC; BD Biosciences Clontech, Palo Alto, Calif.). PCRamplification was performed with AdvanTaq Plus DNA Polymerase reagentsaccording to the Clontech User Manual protocol for two-step cycling. Theforward primer, ERβ GSP6, GAAGCTGGCTCACTTGCTGAACGC (SEQ ID NO:7)combined with a primer designed to the unique exon 7 nucleotidesequence, ERβ X-2, AACTCTCTGCGACAGTGCCATAGA (SEQ ID NO:15) produces anamplicon of 278 bp. Control, G3PDH primers were used to normalize theresults. A single major band of 983 bp was generated using amplificationconditions recommended by the manufacturer.

The relative expression levels of ERβcx2 were determined in these tissuepanels normalized for GAPDH. ERβcx2 mRNA was found to be the mostabundant in testis (FIG. 5). Transcripts were also detected in ovary,small intestine, spleen, and thymus at lower concentrations and weremore easily observed with increasing cycle number. A low level of ERβcx2mRNA expression was observed in fetal thymus (FIG. 5).

Example 6 Relative Abundance of Estrogen Receptor Isoforms

RT-PCR was performed on human testis total RNA. Reverse transcriptionwas performed with Superscript (Invitrogen, Carlsbad, Calif.).Amplification was performed by two-step PCR (denaturing at 95° C. for 30seconds, annealing at 70° C. for 1.5 minutes) using the following primerpairs: hERα-5′, CATCTGGGATGGCCCTACTGCA (SEQ ID NO:18),hERα-3′CATACTTCCCTTGTCATTGGTACTGGCCA (SEQ ID NO:19); hERβ-5′AACTTGGAAGGTGGGCCTGGT (SEQ ID NO:20), hERβ-3′ACCATTCCCACTTCGTAACACTTCCGAA (SEQ ID NO:21); hERβcx2-5′CAGCCTGTTCGACCAAGTGC (SEQ ID NO:22) and hERβcx2-3′GTTAAACTCTCTGCGACAGTGCCATAGAC (SEQ ID NO:23).

Aliquots of the PCR reactions were analyzed at 30 cycles prior to theamplification plateau and again at 35 cycles (FIG. 6).

Example 7 Functional Characterization of ERβcx2 Cell Culture andTransient Transfection

The estrogen response element (ERE) containing reporter, ERα, and ERβexpression plasmids have been described previously (Bodine, et al J CellBiochem 1997; 65: 368-387 and Bhat et al. J Steroid Biochem Mol Biol1998; 67(3):233-40). The ERβcx2 expression plasmid was constructed bycloning a BstXI/XbaI fragment of the original ERβcx2 clone (ef1-3/pCR2.1) into the ERβ expression plasmid digested with the same restrictionenzymes. The sequence of the clone was confirmed by sequence analysis.An SV40-β gal plasmid (Promega, Madison, Wis.) was used to normalizetransfections.

Cells were transfected with lipofectamine 2000 and incubated in thepresence or absence of estrogen for 24 hrs. Luciferase expression wasmonitored in the cell lysates. Transient transfections were performed inHepG2 cells maintained in DMEM supplemented with 10% fetal bovine serum.Twenty-four hours prior to transfection, cells are seeded in phenolred-free (PRF) DMEM containing 10% charcoal-stripped serum (csFBS), intocollagen-coated 6 well plates (Biocoat, Becton Dickenson). Cells (6×10⁵)were transfected with 2-4 μg of receptor or reporter plasmids usingLipofectamine or Lipofectamine 2000 reagent with OPTI-MEM mediaaccording to the manufacturer's protocols (Invitrogen, Carlsbad,Calif.). After 6 hrs, the medium was replaced with 10% csFBS in PRF DMEMcontaining 10⁻⁶ μM 17b-estradiol (E2) or ethanol vehicle. 24 hrs later,cells were and luciferase activity was quantitated in an automated ML1000 Luminometer (Dynex Technologies, Chantilly, Va.,). β-galactosidaseactivity was measured in cell lysates to normalize for differences intransfection efficiency. Preparative transfections for Western analysiswere performed in 100 mm dishes and required volumes and masses to bemultiplied by a factor of 6.

To define the potential functional properties of the ERβcx2 isoform, invitro assays were performed utilizing expression plasmids for variousestrogen receptor isoforms and an ERE-tk reporter construct. In arepresentative experiment (FIG. 7), ligand-activated transcription ofthe ERE-reporter was induced 23-fold by ERα. The ERβ expressionconstruct activated transcription 8.0 fold upon stimulation with E2. ERβis be a weak transcriptional activator and consistently shows lowerefficiency compared with ERβ activity in these assays. By contrast inthese same experiments, luciferase activity was unchanged for the DNAconstruct expressing ERβcx2 and the negative control pcDNA3.0. Thus itappears that, substitution of the c-terminal sequence in ERβcx2 disruptsthe LBD and generates, not unexpectedly, an isoform functionallyinactive for transactivation from a traditional estrogen receptorresponse element.

ImmunoBlot Analysis of Transiently Expressed ERα, ERβ, and ERβcx2

To confirm that protein was being expressed from the ERβcx2 DNAconstruct, immunoblot analysis was performed on cell lysates fromduplicate transfections.

Samples of whole cell lysates (generally 10-25 mg protein) were mixedwith 2×SDS loading buffer denatured and electrophoresed on 10-20% SDSpolyacrylamide gradient gels. Separated proteins were transferred toPVDF membranes (NOVEX) for immunodetection with the ECL detection system(Amersham Pharmacia Biotech, Piscataway, N.J.). A polyclonal anti-humanERα antibody; HC-20 (Panvera, Madison, Wis.), was used to detect the ERαisoform. ERβ and the ERβcx2 fusion were revealed with EF-304 polyclonal(WO99/07847) (LBD; amino acids 247-530). The ERβcx2 fusion is detectedby this polyclonal ERβ antibody despite containing only a portion of theLBD (amino acids 247-469).

The immunoblot assays were performed briefly as follows. PAGE gels wereelectro-blotted (Biorad, Hercules, Calif.) and the filters were blockedfor 2 hrs with 5% dry milk in PBS+0.3% Tween (PBS-T). A 1:1000 (ERβ) or1:4000 (ERβ) dilution of the primary antibody was added to the 5% drymilk in PBS-T for 2-18 hours. Following 3×10 min PBS-T washes, theprimary staining was detected by incubating the filter with a 1:3000dilution of donkey anti-rabbit horseradish peroxidase-conjugated IgG for1 hr in PBS-T. Following 3×PBS-T washes, filters were developed with theECL substrate as recommended (Amersham Pharmacia Biotech, Piscataway,N.J.) and the blots were exposed to Hyperfilm-MP (Amersham PharmaciaBiotech, Piscataway, N.J.).

The polyclonal antibody specific for ERα recognized a single band ofappropriate MW (65 Kd) in both the transfected cells and for therecombinant ERα positive control (FIG. 8, panel A). The second antibodydeveloped to the LBD of ERβ (amino acids 247-530) visualized ERβ in thetransfected cells as well as the recombinant protein. The recombinantbeta protein was significantly overloaded and the higher MW bandpotentially represents aggregated protein in this analysis. ERβcx2 whichis truncated in the LBD, was nonetheless recognized by the ERβpolyclonal as a protein of an appropriately smaller size in transfectedcell lysates (FIG. 8, panel B). Furthermore, since it is likely that notall the epitopes exist in the truncated variant, the signal intensityfor transiently expressed ERβcx2 relative to ERβ may be diminished. Thedata suggests that the ERβcx2 protein is expressed in our transfectionexperiments.

ERαcx2 Isoform Shows Dominant Negative Activity.

Cotransfection experiments were performed to demonstrate potentialdominant negative effects of the ERβcx2 variant on estrogen signaling.In these experiments, the ERE-tk reporter was cotransfected with eitherERα or ERβ and increasing amounts of the ERβcx2 expression plasmid.Luciferase acitivity was measured in 48 hrs. As shown in FIG. 9, ERαinduces transcription maximally with E2 ligand activation. As increasingamounts of the ERβcx2 expression construct are added luciferase activityis diminished. Cotransfection experiments performed with ERβ expressionplasmid demonstrate a similar dominant negative effect on the ERβsignaling pathway. As shown in FIG. 10, ERβ induces transcriptionmaximally with E2 ligand activation and as increasing amounts of theERβcx2 expression construct are added, signaling through ERβ is reduced.In summary therefore, ERβcx2 appears to have the potential to inhibitboth ER signaling pathways, possibly by the generation oftranscriptionally inactive ER's through a non-productiveheterodimerization mechanism. In contrast to the previously publishedERβcx, which showed significant dominant negative activity against ERαtransactivation only, ERβcx2 demonstrates activity for both isoformspotentially. These studies provide the first evidence of a dominantnegative regulator of the ERβ isoform specifically.

1.-6. (canceled)
 7. A polypeptide encoded by an isolated nucleic acidfragment selected from the group consisting of: (a) an isolated nucleicacid fragment encoding SEQ ID NO:1; (b) an isolated nucleic acidfragment encoding an amino acid sequence having at least 95% identitywith the SEQ ID NO:1; (c) an isolated nucleic acid molecule thathybridizes with the isolated nucleic acid fragment of (a) underhybridization conditions of 6×SSC (1M NaCl), 45 to 50% formamide, 1% SDSat 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.; and (d) anisolated nucleic acid fragment that is complementary to (a), (b) or (c).8. The polypeptide of claim 7 as set forth in SEQ ID NO:1.
 9. Thepolypeptide of claim 7 further characterized by estrogen receptordominant negative activity.
 10. The polypeptide of claim 7 furthercharacterized by ERβ dominant negative activity.
 11. The polypeptide ofclaim 7 wherein the polypeptide comprises SEQ ID NO:3.
 12. A polypeptidecomprising SEQ ID NO:3 further characterized by estrogen receptordominant negative activity.
 13. A polypeptide comprising SEQ ID NO:3further characterized by ERβ dominant negative activity. 14.-27.(canceled)
 28. A method of obtaining a nucleic acid fragment encodingthe polypeptide of claim 7, the method comprising: (a) probing a genomiclibrary with all or a portion of a nucleic acid fragment as set forth inSEQ ID NO:2; (b) identifying a DNA clone that hybridizes with thenucleic acid fragment of step (a); and (c) determining the sequence ofthe nucleic acid fragment that comprises the DNA clone identified instep (b).
 29. (canceled)